Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

Kathryn Geraghty , Shuai Chen , Jean E Harthill , Adel F Ibrahim , Rachel Toth , Nick A Morrice , Franck Vandermoere , Greg B Moorhead , D Grahame Hardie , Carol Mackintosh (1)

1 College of Life Sciences

AS160 mediates insulin-stimulated GLUT4 translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography we found that binding of AS160 to 14-3-3 isoforms in HEK293 cells was induced by insulin- like growth factor 1 (IGF1), epidermal growth factor (EGF), phorbol ester (PMA), and to a lesser extent by AICAR. AS160/14-3-3 interactions were stabilised by chemical cross-linking, and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666, and Ser751) were phosphorylated differentially in response to IGF1, EGF, PMA and AICAR. The binding of 14-3-3s to HA-AS160 was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC kinases RSK1, SGK1 and PKB displayed distinct signatures of AS160 phosphorylation in vitro: All three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK preferentially phosphorylated Ser588, with lesser phosphorylation of other sites. In cells, the IGF1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI 3-kinase inhibitors, while the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and use of AICAR in HEK293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF1-induced phosphorylation of the novel phosphoSer666-Pro site was suppressed by AICAR, and by combined mutation of a TOS-like sequence (FEMDI) and rapamycin. Thus, while AS160 is a common target of insulin, IGF1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

Mots clés: Life Sciences

Kathryn Geraghty, Shuai Chen, Jean E Harthill, Adel F Ibrahim, Rachel Toth, et al.. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. Biochemical Journal, 2007, 407 (2), pp.231-241. ⟨10.1042/BJ20070649⟩. ⟨hal-00478803⟩